The highly conserved, ATP-independent chaperone Hsp33 specifically protects bacteria against oxidative stress conditions that lead to protein unfolding. We found that Hsp33 uses the oxidation status of four cysteine residues to control its chaperone activity, making Hsp33 the first redox-regulated chaperone known. By studying the mechanism of Hsp33 action, we realized that activation of Hsp33 involves large-scale conformational rearrangements in the chaperone, essentially converting the complete C-terminal redox switch domain of Hsp33 into an intrinsically disordered protein. Intriguingly, stress-specific activation by partial unfolding has been recently reported for other energy-independent chaperones as well, suggesting that this mechanism may represent a new paradigm in the field of chaperones and intrinsically disordered proteins. We will now combine mutational, biochemical, and structural tools to elucidate the precise working mechanism of Hsp33 with the goal of determining the role that intrinsic disorder plays in chaperone function. Our proposed studies will test a model in which intrinsically disordered chaperones, like Hsp33, utilize fully reversible order to- disorder transitions to control substrate binding and release. I collaboration with Dr. Lewis Kay we will make use of Hsp33's relatively small size, its ability to form very stable complexes with well-characterized substrate proteins and its amenability to NMR, to monitor, at atomic resolution, how intrinsically disordered chaperones interact with substrate proteins to facilitate their refolding. In addition, we will follow up on our recent discovery that Hsp33 not only protects bacteria against the potent antimicrobial hypochlorous acid (i.e., bleach) but also dramatically increases bacterial resistance to bile salts, the first ine of defense used to limit bacterial colonization in the mammalian intestine. Our proposed studies will unravel the mechanism by which these antimicrobials affect bacteria and the protective role that Hsp33 plays in this process. In summary, our studies will provide an important opportunity to understand, in molecular detail, how chaperones like Hsp33 select, bind and impact their substrate proteins. Together with the analysis of Hsp33's role in bleach and bile salt resistance, these results will facilitate the development of novel antimicrobial strategies. PUBLIC HEALTH RELEVANCE: The mammalian host defense produces high levels of oxidants, such as bleach, to kill off invading microorganism. Bacteria defend themselves by using the redox-regulated chaperone Hsp33, which, specifically activated by the presence of these oxidants, protects bacterial proteins against stress-induced unfolding and enhances bacterial stress resistance. We will now elucidate the mechanisms by which Hsp33 binds proteins under oxidative stress conditions, and releases them for efficient refolding once non-stress conditions are restored.